AN INVESTIGATION OF ARSENIC IN BIOLOGICAL SAMPLES FROM UNEXPOSED

313 Pages·2006·2.22 MB·English

AN INVESTIGATION OF ARSENIC IN BIOLOGICAL
SAMPLES FROM UNEXPOSED VOLUNTEERS IN
THE UK

Eid Ibrahim Brima

A thesis submitted to De Montfort University in partial
fulfilment of the requirements of the degree of Doctor of
Philosophy

Faculty of Health and Life Sciences
School of Allied Sciences
De Montfort University Leicester
June 2007
Acknowledgements

The contribution of my Mum Batoul and my Dad Ibrahim cannot be
underestimated, without their love and prayer I would never have made it this far.
Thanks due to De Montfort University (DMU) for granting me a PhD
studentship. My supervisors Dr Parvez I. Haris, Dr Chris F. Harrington and Professor
Richard O. Jenkins for their help and advice through this project.
Thanks to all people who provided access to IC-PMS to make this project
possible: Michael Watts, British Geological Survey, Keyworth; Dave Polya, Andrew
Gault and Paul Lythgoe, The University of Manchester; Gillian Greenway and Gareth
Pearson, The University of Hull.
I would like to thank the chemical store team at DMU: Chris Warrington,
Suzan, and Nazmin, who were always ready to help.
Finally, this project would not have been possible without the help and
understanding of over 93 volunteers who provided me with biological samples ranging
from urine, hair and fingernails. I am particularly indebted to the fasting volunteers
who provided me with urine samples twice in a single day (both in the morning and
evening).

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Abstract
This thesis describes studies on the analysis of arsenic (As) in human biological
samples, mainly urine but also hair and fingernails using inductively coupled plasma
mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry
(GF-AAS). The relationship between ethnicity and arsenic metabolism was investigated
for the first time for a population in the United Kingdom. This investigation has been
carried out through comparative analysis of arsenic in human urine, hair and fingernails
in volunteers from three different ethnic groups (Whites, Asians and Somali Black-
Africans) who are only exposed to background levels of arsenic. Results obtained with
63 volunteers showed ethnic differences in urinary arsenic excretion as well as
differences in arsenic levels in fingernail samples. The averages of total arsenic levels
for the Somali Black-Africans (urine 7.2 µg/g creatinine; fingernails 723 µg/kg) are
significantly (P< 0.05) different from both the Asians (urine 20.6 µg/g creatinine;
fingernails 153.9 µg/kg) and Whites (urine 24.5 µg/g creatinine; fingernails 177.0
µg/kg). The Somali group also shows a higher percentage (50%) of dimethylarsinate
(DMA) and a lower percentage (48%) of arsenobetaine (AB), compared to Asians
(16% DMA and 83% AB) and Whites (22% DMA and 77% AB).
The effect of fasting on urinary arsenic species distribution was also
investigated by monitoring urine samples from 29 Ramadan fasting volunteers, with
each volunteer providing a sample at the beginning (RF1) and at the end (RF2) of an
approximately 12 hours fast. The results obtained showed the frequency of MA
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detection for RF2 was 12 and 2-fold higher than for the non-fasting and RF1
groups, respectively. This suggests fasting may alter the pattern of arsenic metabolism
and excretion. However, there was no significant difference (P> 0.05) in the average of
total level of arsenic for RF1 (18.3 µg/g creatinine) and RF2 (17.7 µg/g creatinine).
A relationship between excretion of arsenic and selenium in individuals exposed
to background levels of arsenic and selenium was investigated through analysis of urine
samples from 93 volunteers from Leicester, UK. A positive correlation between
arsenic and selenium was found and the As:Se ratio was 0.7 ± 0.4. The intra-individual
variation of As:Se ratio does not alter significantly over time, as determined by
monitoring urine samples from a volunteer over a period of one year. Furthermore,
within a single day, with urine samples collected at the beginning and after a 12-hour
fast, the As:Se ratio was found to be similar (0.7 ± 0.5). These findings suggest a close
relationship between these two metalloids, the biological significance of which needs to
be explored in the future.

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Contents
ACKNOWLEDGEMENTS.................................................................................. I
ABSTRACT....................................................................................................... II
CONTENTS......................................................................................................IV
PUBLICATIONS ............................................................................................XIII
LIST OF FIGURES..........................................................................................XV
LIST OF TABLES........................................................................................XVIII
LIST OF ABBREVIATIONS...........................................................................XXI
CHAPTER 1...................................................................................................... 1
INTRODUCTION............................................................................................... 1
1.1 Arsenic Chemistry....................................................................................................1
1.2 Arsenic occurrence...................................................................................................5
1.2.1 Natural occurrence of arsenic...........................................................................6
1.2.1.1 Arsenic in the earth’s crust.......................................................................6
1.2.1.2 Arsenic in soil...........................................................................................6
1.2.1.3 Arsenic in water........................................................................................7
1.2.1.4 Arsenic in food.........................................................................................9
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1.2.1.5 Arsenic in Plants.........................................................................................9
1.2.1.6 Arsenic in the human body.....................................................................10
1.2.2 Anthropogenic occurrence of arsenic.............................................................11
1.3 Arsenic toxicity.......................................................................................................12
1.4 Arsenic metabolism in humans.............................................................................16
1.4.1 Arsenic metabolism in relation to various parameters...................................18
1.4.1.1 Definition of exposed and unexposed volunteers...................................20
1.4.1.2 Arsenic metabolism as a function of exposure.......................................20
1.4.1.3 Understanding arsenic metabolism through ingestion studies...............22
1.4.1.4 Arsenic metabolism as a function of ethnicity/genetics.........................23
1.4.1.5 Homocysteine and folate........................................................................23
1.4.1.6 Selenium.................................................................................................24
1.5 Biomonitoring of exposure to arsenic in humans................................................25
1.5.1 Arsenic determination in humans’ biological samples...................................26
1.5.2 Urinary arsenic analysis....................................................................................27
1.5.2.1 Collection and storage of urine...............................................................28
1.5.2.2 Arsenic speciation versus total arsenic analysis in urine.......................29
1.5.2.3 Spectroscopic techniques for arsenic determination in human urine.....30
1.5.3 Arsenic determination in human hair and fingernail......................................32
1.6 Aims of the thesis....................................................................................................33
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1.7 Summary ................................................................................................................34
1.8 References ...............................................................................................................35
CHAPTER 2.................................................................................................... 51
INSTRUMENTATION, SAMPLE PRE-TREATMENT, EXPERIMENTAL
PROCEDURES AND METHOD DEVELOPMENT......................................... 51
SECTION 1..................................................................................................... 52
2.1 Theoretical background to Instrumentation, Sample Pre-treatment and
Chromatography..........................................................................................................52
2.1.1 Graphite furnace atomic absorption spectrometry (GF-AAS)......................52
2.1.2 Inductively coupled plasma mass spectrometry (ICP-MS)............................56
2.1.3 Sample pre-treatment and chromatographic separation for arsenic in human
urine ................................................................................................................65
2.1.3.1 Sample filtration........................................................................................65
2.1.3.2 Sample digestion......................................................................................65
2.1.3.3 Chromatographic separation for arsenic in human urine..........................66
SECTION 2..................................................................................................... 68
2.2 Experimental procedures and method development..........................................68
2.2.1 Methodology...................................................................................................69
2.2.1.1 Methodological steps for arsenic determination in biological samples..69
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2.2.1.2 Ethical approval, urine sample collection and preparation ....................70
2.2.1.3 Determination of creatinine....................................................................71
2.2.2 Method development for total arsenic using GF-AAS...................................74
2.2.2.1 Chemical modifier selection....................................................................74
2.2.2.2 Dilution factor selection.........................................................................76
2.2.2.3 Recovery test for the modifiers and dilution factors..............................76
2.2.2.4 The modifier of choice...........................................................................77
2.2.2.5 Limit of detection and limit of quantification........................................78
2.2.2.6 Method validation...................................................................................78
2.2.2.7 Hair and fingernail collection, pre-treatment and total arsenic
determination..........................................................................................................79
2.2.3 Establishing a method for arsenic speciation in human urine using HPLC-GF-
AAS (off-line)............................................................................................................80
2.2.4 Method development for total arsenic and selenium in human urine using
ICP-MS ................................................................................................................85
2.2.4.1 Internal standard selection.....................................................................85
2.2.4.2 Chloride interference..............................................................................86
2.2.4.3 Determination of total arsenic in urine samples and method validation87
2.2.4.4 Determination of total selenium in urine samples and method validation
................................................................................................................92
2.2.5 Method development for arsenic speciation in human urine using
HPLC- ICP-MS..........................................................................................................95
2.2.5.1 Chromatographic conditions..................................................................96

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2.2.5.2 Speciation analysis of arsenic in urine samples and method
validation................................................................................................................96
2.2.6 Statistical analysis........................................................................................106
2.3 Summary discussion of modified/developed methods.......................................110
2.4 References .............................................................................................................112
CHAPTER 3.................................................................................................. 120
ETHNICITY AND ARSENIC METABOLISM................................................. 120
3.1 Introduction..........................................................................................................120
3.2 Experimental.........................................................................................................123
3.2.1 Study group..................................................................................................123
3.2.1.1 Time delay between urine samples collection, storage and analysis....124
3.2.1.2 Urine samples collection, storage and preparation...............................126
3.2.1.3 Dates of collection and measurements of urine samples from the three
ethnic groups........................................................................................................127
3.2.2 Chemicals and reagents................................................................................128
3.2.3 Instrumentation.............................................................................................128
3.2.4 The procedure to determine the effect of seafood consumption on the
background level of total urinary arsenic................................................................129
3.2.5 Quality control and method validation.........................................................129
3.2.6 Determination of creatinine..........................................................................129
3.2.7 The procedure to determine the effect of storage time on arsenic compound
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stability ..............................................................................................................130
3.2.8 Determination of total arsenic in urine samples.........................................130
3.2.9 Speciation analysis of arsenic in urine samples........................................130
3.2.10 Collection, pre-treatment and total arsenic determination of arsenic in
hair and fingernail sampples.....................................................................................131
3. 2.11 Statistical analysis.....................................................................................131
3. 3 Results ..............................................................................................................131
3. 3. 1 Analysis of urine samples............................................................................131
3.3.1.1 Effect of seafood consumption on the background of urinary arsenic
level ..............................................................................................................132
3.3.1.2 Relationship between storage conditions and the stability of total
arsenic in urine samples........................................................................................134
3.3.1.3 Relationship between storage conditions and the stability of arsenic
species in urine samples………………...................................................................135
3.3.1.4 Representative chromatograms for speciation of arsenic in urines amples
..............................................................................................................136
3.3.1.5 Concentration of total arsenic and arsenic species before creatinine
adjustment among the three ethnic groups...........................................................139
3.3.1.6 Concentration of total arsenic and arsenic species after creatinine
adjustment among the three ethnic groups...........................................................141
3.3.1.7 Arsenic concentrations in urine samples...............................................146
3. 3. 2 Analysis of hair and fingernail samples....................................................151
3.4 Discussion.............................................................................................................158
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